DESCRIPTION: The ubiquitin-proteasome pathway is the predominant mechanism for the turnover of short-lived proteins in eukaryotic cells. The role of ubiquitin in proteolysis is that of a signal; ubiquitination confers substrate recognition by the proteasome. In this pathway, the principal signal is a polyubiquitin chain linked by Lys48-Gly76 isopeptide bonds between successive ubiquitins. The interaction of this chain with proteasomal recognition factors ultimately determines the stability of a large fraction of intracellular proteins. Previous work by the principal investigator and others has shown that assembling ubiquitin into such a chain potentiates the recognition of the ubiquitin proteolytic signal, but the molecular basis of this effect is poorly understood. In this proposal, the principal investigator will seek a detailed understanding of the recognition of polyubiquitin signal. This goal will be achieved by delineating the molecular features of the chain which are important for its recognition, by identifying and characterizing novel proteasomal components which are responsible for recognizing the chain, and by conducting mechanistic studies of purified proteasomes, so as to elucidate the coupling between polyubiquitin signal recognition and substrate turnover. Studies will test the novel hypothesis that assembling ubiquitin into a Lys48-linked chain creates a three dimensional signal that is recognized by specific factors within the regulatory model of the 26S proteasome. Besides targeting substrates to the proteasome, ubiquitination can signal alternate fate. The ability of the cell to distinguish among several fates that are potentially available to ubiquitinated proteins requires that there exist mechanisms for the selective recognition of these species, but these mechanisms remain to be defined. The discovery of polyubiquitin raises the possibility that alternatively-linked polyubiquitin chains serve to diversify the signaling functions of ubiquitin. This hypothesis will be treated in the proposed research through analysis of the structure and recognition of polyubiquitin chains that are linked through Lys63.